CN111777664A - Walnut meal antioxidant peptide YFW and preparation method and application thereof - Google Patents
Walnut meal antioxidant peptide YFW and preparation method and application thereof Download PDFInfo
- Publication number
- CN111777664A CN111777664A CN202010722989.2A CN202010722989A CN111777664A CN 111777664 A CN111777664 A CN 111777664A CN 202010722989 A CN202010722989 A CN 202010722989A CN 111777664 A CN111777664 A CN 111777664A
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- Prior art keywords
- polypeptide
- walnut
- antioxidant
- solution
- walnut pulp
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Links
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- 238000002360 preparation method Methods 0.000 title claims abstract description 6
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- 240000007049 Juglans regia Species 0.000 title description 2
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- 108700016172 Glutathione peroxidases Proteins 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
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- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
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- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
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- 229910052742 iron Inorganic materials 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
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- 230000000050 nutritive effect Effects 0.000 description 1
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- 235000020636 oyster Nutrition 0.000 description 1
- 235000021118 plant-derived protein Nutrition 0.000 description 1
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 1
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- 235000019165 vitamin E Nutrition 0.000 description 1
- 229940046009 vitamin E Drugs 0.000 description 1
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- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 239000008170 walnut oil Substances 0.000 description 1
- 235000021119 whey protein Nutrition 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/001—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from waste materials, e.g. kitchen waste
- A23J1/005—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from waste materials, e.g. kitchen waste from vegetable waste materials
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
- A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
- A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
- A23J3/348—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of proteins obtained from waste materials
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/90—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in food processing or handling, e.g. food conservation
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Nutrition Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- Toxicology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Plant Substances (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention belongs to the technical field of deep processing of food, and particularly relates to an antioxidant polypeptide extracted from walnut meal and a preparation method and application thereof. The antioxidant peptide is Tyr-Phe-Trp (the C end is Trp, and the N end is Tyr). In order to obtain a product containing the antioxidant peptide, the walnut pulp is used as a raw material, alkaline protease is used for enzymolysis, enzymolysis liquid is subjected to ultrafiltration separation to obtain walnut pulp polypeptides with different molecular weight ranges (0-3KDa, 3-5KDa, 5-10KDa and >10KDa), glucan gel chromatography G-25 purification is continuously performed, and the obtained YFW polypeptide has high antioxidant activity, is beneficial to removing free radicals in a body and has antioxidant activity.
Description
The technical field is as follows:
the invention belongs to the technical field of food deep processing, and particularly relates to an antioxidant peptide extracted from walnut meal and a preparation method and application thereof.
Background art:
reactive oxygen Radicals (ROS) such as superoxide anions, hydroxyl radicals, and hydrogen peroxide are generally generated in the metabolic processes of organisms. The antioxidant defense system of the human body, such as endogenous antioxidant enzymes (including superoxide dismutase, catalase, glutathione peroxidase, and the like) and antioxidant small molecules (including vitamin C, vitamin E, glutathione, and the like), plays an important role in eliminating active oxygen and preventing cell damage. However, excessive production of ROS can cause oxidative damage, which in turn can induce oxidation of biomolecules such as DNA, RNA, and membrane lipids, leading to various diseases such as cancer, atherosclerosis, diabetes, and cardiovascular diseases, as well as aging. Therefore, there is a need to ingest sufficient antioxidants to prevent or slow the oxidative stress caused by ROS. In food systems, the formation of toxic compounds such as lipid peroxides and lipid radicals caused by oxidative deterioration of foods is responsible for the degradation of food quality and safety. In order to maintain the flavor, color and nutritional value of food products, it is necessary to inhibit lipid peroxidation by adding antioxidants. In the food industry, synthetic antioxidants have been used in food products to prevent deterioration of oxidation, however, due to their potential health hazards, the use of synthetic antioxidants is strictly regulated. Therefore, the search for safe natural antioxidants is an important issue in the technical field of food science.
In recent years, antioxidant polypeptides have attracted much attention due to their advantages of wide sources, small molecular weight, easy absorption, safety, no toxicity, and the like. The research range of people on the anti-oxidation peptide is wide, such as: soybean peptide, oyster peptide, egg protein peptide, whey protein peptide and the like, and in addition, some more specific antioxidant peptides such as: recombinant Trx-TAT-hMsrA protein and the like developed based on methionine sulfoxide reductase A (an antioxidant enzyme) all show stable, safe and effective antioxidant and anti-aging functions. The natural reducing peptide can reduce the occurrence probability of related diseases such as oxidation, aging and the like, and therefore, the natural reducing peptide is often used for research and development of anti-aging health-care food and cosmeceutical. Compared with artificially synthesized reducing agents, the reducing peptide obtained by enzymolysis of the plant protein is more easily absorbed by human bodies, and has no toxic or side effect. In the aspect of food, the antioxidant peptide is directly added into the food as a raw and auxiliary material, so that the oxidative deterioration of the food can be effectively prevented or slowed down, the quality of the food is improved, and the shelf life of the food is prolonged; in the cosmetic and pharmaceutical industries, antioxidant peptides are directly added into cosmetics or medicines as important raw materials, and can effectively prevent and treat various diseases caused by free radicals generated by oxidation reaction and slow down the aging speed of the body. At present, antioxidant peptide emulsion, antioxidant peptide mask, antioxidant peptide medicine, etc. are also gradually appearing in the market.
Walnut (Juglans regia L.) is one of common nut crops in the market and has rich nutritional value and medicinal efficacy. A large amount of by-product walnut meal is produced after walnut oil extraction and is usually sold at a low price by feed, so that not only is walnut protein resource wasted, but also the development of the walnut industry is seriously hindered. The biological enzymolysis technology is used as a green biotransformation means, which is helpful for improving the bioavailability of the substrate on one hand, and decomposing the walnut meal protein into small molecular compounds which are more beneficial to the absorption of the human body on the other hand, thereby increasing the functional activity and improving the nutritive value.
The research uses cold-pressed walnut pulp as a raw material, prepares walnut polypeptide by a biological enzymolysis method, separates and purifies walnut pulp protein enzymolysis liquid by ultrafiltration, sephadex chromatography and other methods, measures ABTS free radical scavenging capacity, screens out polypeptide components with high antioxidant activity, performs liquid chromatography on the purified walnut pulp polypeptide, determines the amino acid sequence of the walnut pulp polypeptide, artificially synthesizes antioxidant polypeptide, and detects antioxidant activity (DPPH free radical scavenging capacity, ABTS free radical scavenging capacity, hydroxyl free radical scavenging capacity and iron ion reduction capacity).
The invention content is as follows:
in order to solve the technical problems, the invention firstly provides an antioxidant peptide extracted from walnut dregs, wherein the amino acid sequence of the antioxidant peptide is Tyr-Phe-Trp (the C end is Trp, and the N end is Tyr).
The invention also provides a method for obtaining the polypeptide, which comprises the following steps:
the invention uses walnutsThe method comprises degreasing with petroleum ether, extracting protein from defatted walnut cake by alkali extraction and acid precipitation, performing enzymolysis with alkaline protease, ultrafiltering and separating the enzymolysis solution to obtain protein with different molecular weight ranges (0-3KDa, 3-5KDa, 5-10KDa, etc.),>10KDa) of walnut pulp polypeptides, detecting ABTS free radical scavenging capacity of the walnut pulp polypeptides to obtain walnut pulp polypeptides with high ABTS free radical scavenging capacity after ultrafiltration, continuing to carry out sephadex chromatography G-25 purification, detecting ABTS free radical scavenging capacity of the walnut pulp polypeptides in the same way to obtain walnut pulp polypeptides with high antioxidant activity after purification, and then adopting an EASY nano-LCsytem liquid chromatograph, C18(75 μm × 100mm, 3 μm), connecting with LTQ orbitrap velos pro mass spectrometer in series, determining the structure of the walnut pulp polypeptide, then artificially synthesizing the corresponding polypeptide, and detecting DPPH free radical scavenging capacity, ABTS free radical scavenging capacity, hydroxyl free radical scavenging capacity and reducing power, thereby obtaining the short peptide sequence with high antioxidant activity in the walnut pulp polypeptide.
The invention also provides the application of the antioxidant peptide and an antioxidant product containing the antioxidant peptide.
Has the advantages that:
the invention provides a novel antioxidant peptide which has obvious antioxidant effect, adopts walnut pulp as a raw material, and has low production cost and high economic benefit.
Based on test results, the ABTS free radical clearance rate determination result of the walnut pulp polypeptide after ultrafiltration and enzymolysis is that the ABTS free radical clearance activity of the walnut pulp polypeptide with the molecular weight of 0-3KDa is highest, and the synthesized short peptide sequence YFW is proved to have higher antioxidant activity. The invention is beneficial to removing free radicals in vivo and has antioxidant activity.
Description of the drawings:
FIG. 1 shows the scavenging effect of polypeptides of different molecular weights on ABTS free radicals;
FIG. 2 Sephadex chromatography purification diagram;
FIG. 3 shows the scavenging effect of small molecule polypeptides on ABTS free radicals after purification by Sephadex chromatography;
FIG. 4 is a total ion flow diagram of a walnut meal polypeptide;
FIG. 5 is a liquid mass spectrum of a walnut cake polypeptide Tyr-Phe-Trp;
FIG. 6 shows that the ABTS free radical is eliminated by artificially synthesizing small molecular polypeptide YFW;
FIG. 7 shows that the synthetic small molecule polypeptide YFW has DPPH free radical scavenging effect;
FIG. 8 shows that the synthesized small molecular polypeptide YFW has effect of scavenging hydroxy free radicals;
FIG. 9 shows the reducing power of artificially synthesized small molecule polypeptide YFW.
The specific implementation mode is as follows:
in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. The specific embodiments described herein are merely illustrative of the present patent and are not to be construed as limiting the invention
Example 1: extraction method of walnut meal polypeptide
(1) Walnut meal degreasing:
pulverizing walnut cake, sieving with 40 mesh sieve, extracting with petroleum ether at a material-to-liquid ratio (w/v) of 1: 5 for 2 hr, filtering, collecting residue, continuously extracting for 3 times, volatilizing organic solvent from the residue in a fume hood to obtain walnut cake defatted powder, and storing at 4 deg.C.
(2) Extracting walnut protein by alkali dissolution and acid precipitation
Accurately weighing 10g of walnut meal defatted powder in a 400mL beaker, adding 200mL of distilled water, adjusting the pH to 10.0, stirring on a magnetic stirrer for 5min, then carrying out ultrasonic treatment for 20min, immediately taking out after the ultrasonic treatment is finished, standing for 1h, centrifuging at 6000r/min for 20min, and taking the supernatant for later use. Adjusting pH to 4.5 with 1mol/L HCl, standing for 1h, centrifuging at 6000r/min for 20min to obtain walnut cake protein precipitate, adjusting pH to 7.0 with 1mol/L NaOH to obtain walnut cake protein, and vacuum freeze drying for use.
(3) Alkaline protease treatment
Mixing the prepared walnut pulp protein freeze-dried powder with distilled water at a feed-liquid ratio (w/v) of 1: 20, and pre-boiling for 30min at 90 ℃. After the pre-cooking is finished, when the temperature is reduced to 60 ℃, the pH value is adjusted to 10.0 by using 1mol/L NaOH solution, and 5% (w/w) of alkaline protease is added to hydrolyze the walnut pulp protein solution for 5 hours. The pH of the reaction was maintained at 10.0 throughout the hydrolysis by the addition of 1mol/L NaOH solution. After the enzymolysis is finished, boiling for 10min to stop the enzymatic reaction, adjusting the pH to the isoelectric point of the protein by 1mol/L HCl solution to remove macromolecular protein, centrifuging for 20min at 6000r/min, taking supernatant, adjusting the pH to 7.0, and freeze-drying for later use.
(4) Walnut meal polypeptide purification method
Coarse walnut pulp polypeptide → dissolution → ultrafiltration filter → vacuum freeze drying → preparation of small molecular polypeptide solution → sephadex chromatography purification → purified liquid freeze-drying preservation.
Centrifuging the walnut cake protein zymolyte for 10min at 4000r/min, taking supernatant, carrying out microfiltration treatment on the supernatant with the diameter of 0.8 mu m, carrying out interception separation on the supernatant by using a 10KDa ultrafiltration membrane, and reserving concentrated solution; intercepting and separating the filtrate by a 5KDa ultrafiltration membrane, and reserving a concentrated solution; and intercepting and separating the filtrate by using a 3KDa ultrafiltration membrane, and reserving the filtrate and the concentrated solution. Keeping the maximum working pressure of the component at 0.1MPa in the ultrafiltration process, and sequentially preparing walnut pulp polypeptide solution with molecular weight of more than 10KDa, 5-10KDa, 3-5KDa and less than 3 KDa.
Separating the ultrafiltered walnut pulp polypeptide by Sephadex G-25 chromatography. The separation conditions were: the sample loading amount is 5mg/mL, 5mL, the specification of a chromatographic column is 1.6X 60cm, the flow rate: 1mL/min, using distilled water as eluent, and detecting by using an ultraviolet detector, wherein the wavelength is 220 nm.
Example 2 ABTS free radical scavenging ability of post-ultrafiltrate walnut cake polypeptides of different molecular weights
Respectively weighing 5mg of freeze-dried powder of walnut pulp polypeptide solution with molecular weight of more than 10KDa, 5-10KDa, 3-5KDa and less than 3KDa obtained after ultrafiltration in the step (4) of the embodiment 1, and respectively dissolving vitamin C1mg in 5mL of water to prepare polypeptide with concentration of 1mg/mL and vitamin C solution with concentration of 0.2 mg/mL.
The method comprises the following steps of (1) dividing treatment into 5 treatment steps:
treatment 1: 0.1mL of a polypeptide solution with a molecular weight >10 KDa;
and (3) treatment 2: 0.1mL of polypeptide solution with molecular weight of 5-10 KDa;
and (3) treatment: 0.1mL of polypeptide solution with molecular weight of 3-5 KDa;
and (4) treatment: 0.1mL of a solution of a polypeptide having a molecular weight of 3KDa or less;
and (4) treatment 5: the positive control group is 0.1mL of vitamin C solution;
the determination was carried out according to the ABTS free radical scavenging ability determination method, each treatment was repeated three times, and IC was determined and calculated for the different treatments50The value is obtained.
The result is shown in fig. 1, when the concentration of the walnut pulp polypeptide is 1mg/mL, the ABTS free radical clearance rate is gradually reduced along with the increase of the molecular weight, the ABTS free radical clearance rate of the 0-3KDa walnut pulp polypeptide is 37.49%, the ABTS free radical clearance rate of the walnut pulp polypeptide slightly higher than 3-5KDa is 32.43%, but the ABTS free radical clearance rate of the walnut pulp polypeptide is obviously higher than 22.76% and 21.88% of that of the walnut pulp polypeptide with 5-10KDa and >10 KDa;
the ABTS free radical scavenging rate difference of 0-3K, 3-5K, 5-10KDa, walnut pulp polypeptide with >10KDa and vitamin C is obvious, and the ABTS free radical scavenging rate of the 0-3KDa walnut pulp polypeptide is 37.49% at most;
IC of ABTS free radical from different molecular weight polypeptides of Table 150The value is seen that the antioxidant activity of the small molecular polypeptide of 0-3KDa is better. Therefore, the walnut pulp polypeptide has a certain ABTS free radical scavenging capacity, and the activity of the small molecular polypeptide is more remarkable.
TABLE 1 ABTS radical scavenging Capacity of different molecular weight Polypeptides
Example 30-3 kDa Small molecule polypeptide Sephadex chromatography
Separating the ultrafiltered small molecular polypeptide of 0-3KDa by Sephadex G-25 chromatography. The separation conditions were: sample loading 5mg/mL, 5mL, chromatographic column specification of 1.6X 60cm, flow rate: 1mL/min, using distilled water as eluent, and detecting by using an ultraviolet detector, wherein the wavelength is 220 nm.
Performing Sephadex chromatography G-25 on the ultrafiltered small molecular polypeptide of 0-3KDa to obtain three peaks of polypeptide: peak 1F 1, peak 2F 2, and peak 3F 3, the results of which are shown in fig. 2, were subjected to ABTS radical scavengingThe results of the rate measurement are shown in FIG. 3. The ABTS free radical clearance of the component F2 is higher than that of the component F1 and the component F3(P is less than 0.05), and when the concentration is 1mg/mL, the ABTS free radical clearance of the components F1, F2 and F3 is respectively 30.92%, 43.27% and 37.54%. IC of ABTS radical of different composition from Table 250As can be seen, the activity of the purified fraction F2 was higher.
TABLE 2 ABTS radical scavenging Capacity of different fractions after Sephadex chromatography purification
Example 4 liquid mass spectrometry of walnut meal polypeptide
Reductive alkylation
A10 mu L F2 fraction was sampled and 50mmol/L NH was added4HCO3Adding 90 mu L of solution into DTT solution to make the final concentration of the solution be 10mmol/L, and reducing the solution in water bath at 37 ℃ for 4 h; adding IAA solution to make the final concentration 50mmol/L, and reacting for 40min in dark; desalting with self-packed desalting column, and volatilizing solvent in vacuum centrifugal concentrator at 45 deg.C.
Mass spectrometric analysis
Capillary liquid chromatography conditions: pre-column: acclaim
C18，300μm×5mm，5μm，
75 μm × 150mm, Acclaim
C18，3μm，
Detection wavelength: 220 nm; mobile phase A: 0.1% formic acid, 2% acetonitrile; mobile phase B: 0.1% formic acid, 80% acetonitrile; column temperature: 30 ℃; flow rate: 300 nL/min. The liquid chromatography elution gradient is shown in table 3.
TABLE 3 liquid chromatography elution gradient
Mass spectrum conditions:
primary mass spectrum parameters:
Resolution：70000AGC；AGC target：3e6；Maximum IT：40ms；Scan range：100-1500m/z；
secondary mass spectrum parameters:
Resolution：75000；AGC target：1e5；Maximum IT：60ms；Top N：20；NCE/steppedNCE：27；Scan range：50-1500m/z；
fig. 4 is a total ion flow diagram of a walnut pulp polypeptide F2 component, wherein the number of polypeptides of number 2 chromatographic peaks in sephadex chromatography is 606 through liquid mass structure analysis, a polypeptide YFW containing tryptophan at the C-terminal is selected as a research object, and liquid mass spectrograms of the polypeptide YFW are respectively shown in fig. 5.
EXAMPLE 5 synthetic Polypeptides against Oxidation
Artificially synthesizing short peptide YFW, and respectively measuring ABTS, DPPH, hydroxyl radical scavenging ability and reducing power.
FIGS. 6-9 show ABTS, DPPH, hydroxyl radical scavenging and reducing power at polypeptide concentrations of 0.2, 0.4, 0.6, 0.8, 1mM and vitamin C concentrations of 0.04, 0.08, 0.12, 0.16, 0.2mM, respectively. It is known that YFW has a certain antioxidant activity, and the antioxidant activity gradually increases with increasing polypeptide concentration, but is lower than that of vitamin C in the positive control group.
Table 4 shows ABTS, DPPH, IC of hydroxyl radical of artificially synthesized short peptide YFW and vitamin C50Value and reducing power at a polypeptide concentration of 1mM and a vitamin C concentration of 0.2 mM.
TABLE 4 antioxidant Activity of synthetic short peptides YFW
Example 6 method for measuring ABTS radical scavenging ability of the present invention
5mL of 7mmol/L ABTS was dissolvedMixing the solution with 88 mu L of 140mmol/L potassium persulfate solution, and standing for 12-16 h at room temperature in a dark condition to obtain ABTS stock solution. Diluting with 10mmol/L phosphate buffer solution (pH 7.4) to give ABTS working solution with absorbance of 0.7 + -0.02 at 734nm wavelength. Taking 20 mu L of solution to be detected and 280 mu L of ABTS working solution to a 96-hole enzyme label plate, and measuring the light absorption value A at 734nm1The light absorption value of the solution to be detected is replaced by deionized water as A2And vitamin C is used as a positive control. ABTS free radical clearance calculation formula is as follows:
clearance rate ═ a2-A1)/A2×100％。
EXAMPLE 7 method for measuring DPPH radical scavenging ability according to the present invention
Preparing a freeze-dried sample into a solution with the mass concentration of 2.0mg/mL by using ultrapure water, putting 2.0mL into a 10mL test tube, adding 1.0mL of a phosphate buffer solution (pH value of 7.0) with the concentration of 0.2mol/L and 2.0mL of a DPPH radical ethanol (volume fraction of 95% ethanol) solution with the concentration of 0.2mmol/L, uniformly mixing, standing in a dark place at room temperature for 30min, measuring the absorbance at the wavelength of 518nm, and taking vitamin C as a positive control. The clearance calculation formula is as follows:
clearance (%) - (1- (A)1-A2)/A0]×100％
In the formula: a. the1-absorbance of the sample solution + phosphate buffer + DPPH ethanol solution;
A2-absorbance of sample solution + phosphate buffer + 95% volume fraction ethanol solution;
A0-absorbance of ultrapure water + phosphate buffer + DPPH ethanol solution.
EXAMPLE 8 measurement of the scavenging ability of hydroxyl radical according to the present invention
Sequentially adding 2.0mL of 6mmol/L ferrous sulfate solution, 2.0mL of samples with different concentrations and 2.0mL of 6mmol/L hydrogen peroxide solution into a 10mL test tube, shaking uniformly, standing for 10min, adding 2.0mL of 6mmol/L salicylic acid solution, shaking uniformly, standing for 30min, and measuring absorbance A at 510nm510And vitamin C is used as a positive control.
Clearance (%) ═ a0-(Ai-Aj)]/A0×100％
In the formula: a. the0-absorbance of sample-free solution;
Ai-absorbance after addition of sample solution;
Ajabsorbance of samples without addition of salicylic acid solution.
EXAMPLE 9 method for measuring reducing power of the present invention
1mL of the sample solution was added with 1mL of 0.2mol/L phosphate buffer (pH 6.6) and 1mL of 1% potassium ferricyanide (K)3Fe(CN)6) Mixing the solution, performing water bath at 50 ℃ for 20min, adding 1mL of 10% trichloroacetic acid (TCA) solution, mixing, and centrifuging at 10000r/min for 10 min. 1mL of the supernatant was added with 1mL of deionized water and 0.2mL of 0.1% FeCl3Mixing the solution, water bathing at 50 deg.C for 10min, and measuring absorbance at 700nm wavelength. Deionized water was used as a blank control instead of the sample, and vitamin C was used as a positive control.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the patent. It should be noted that, for those skilled in the art, various changes, combinations and improvements can be made in the above embodiments without departing from the patent concept, and all of them belong to the protection scope of the patent. Therefore, the protection scope of this patent shall be subject to the claims.
Claims (3)
1. The antioxidant peptide is YFW, the amino acid sequence is Tyr-Phe-Trp, the C end is Trp, and the N end is Tyr.
2. The method for preparing the antioxidant peptide as claimed in claim 1, which is characterized by comprising the following steps:
the method comprises the steps of using walnut pulp as a raw material, degreasing the walnut pulp by using petroleum ether, extracting protein in the degreased walnut pulp by using an alkali extraction and acid precipitation method, performing enzymolysis by using alkaline protease, performing ultrafiltration separation on enzymolysis liquid to obtain walnut pulp polypeptide with the molecular weight of 0-3KDa, and purifying the walnut pulp polypeptide by using glucan gel chromatography G-25.
3. Use of the antioxidant peptide of claim 1 in the preparation of an antioxidant product.
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| CN113307843A (en) * | 2021-06-15 | 2021-08-27 | 北京林业大学 | Walnut meal anti-inflammatory peptide WSL and application thereof |
| CN113845566A (en) * | 2021-11-08 | 2021-12-28 | 西北大学 | Walnut polypeptide for preventing Alzheimer's disease and application thereof |
| CN114958947A (en) * | 2022-03-25 | 2022-08-30 | 江苏大学 | Preparation and purification identification method of oat antioxidant peptide |
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| CN103497985A (en) * | 2013-09-06 | 2014-01-08 | 华南理工大学 | Walnut protein antioxidant peptides and preparation method thereof |
| CN104293870A (en) * | 2014-09-25 | 2015-01-21 | 汾州裕源土特产品有限公司 | Preparation method of antioxidant walnut polypeptide health product |
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| CN103497985A (en) * | 2013-09-06 | 2014-01-08 | 华南理工大学 | Walnut protein antioxidant peptides and preparation method thereof |
| CN104293870A (en) * | 2014-09-25 | 2015-01-21 | 汾州裕源土特产品有限公司 | Preparation method of antioxidant walnut polypeptide health product |
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| CN113307843A (en) * | 2021-06-15 | 2021-08-27 | 北京林业大学 | Walnut meal anti-inflammatory peptide WSL and application thereof |
| CN113845566A (en) * | 2021-11-08 | 2021-12-28 | 西北大学 | Walnut polypeptide for preventing Alzheimer's disease and application thereof |
| CN113845566B (en) * | 2021-11-08 | 2022-11-01 | 西北大学 | Walnut polypeptide for preventing Alzheimer's disease and application thereof |
| CN114958947A (en) * | 2022-03-25 | 2022-08-30 | 江苏大学 | Preparation and purification identification method of oat antioxidant peptide |
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